This project will focus on the molecular changes in exocytic and lipid secretory pathways observed to be functionally up-regulated during the Stage II transition from pregnancy from pregnancy to lactation in mammary epithelial cells. Our major hypothesis is that differential expression and/or post-translational modifications of a subset of resident of resident Golgi and cytoplasmic lipid droplet (CLD) proteins allow for the amplification of the Golgi and changes in the CLDs in preparation for extensive milk protein and lipid secretion. An unbiased global proteomics analysis (2D-gel electrophoresis/mass spectrometry) will be carried out a multiple time points during lactogenesis to identify the sequence in which endogenous and novel proteins are differentially expressed/modified resulting in Golgi expansion and lipid secretion. A select group of these proteins will be functionally characterized during the transition from pregnancy to lactation. Preliminary studies on RLP30, a novel Golgi matrix-like protein have led us to our current hypothesis that remodeling of the Golgi matrix plays a crucial role in organellar expansion. The mechanism of lipid secretion prosed involves interactions among xanthine oxidoreductase (XOR), butyrophilin (Btn), adipocyte differentiation related protein (ADRP), and a membrane associated thiol-oxidase. These interactions will be investigated at molecular and functional levels and proteomics will be used to identify additional components of the lipid secretion process. These approaches will establish the fundamental framework for future studies of milk secretion.